Cell Division in Escherichia coli

Transcription

1 JOURNAL OF BACTERIOLOGY, Nov. 1993, P /93/ $()2.00/0 Copyright C) 1993, American Society for Microbiology Vol. 175, No. 21 Antisense Transcription of the ftsz-ftsa Gene Junction Inhibits Cell Division in Escherichia coli SUSAN J. DEWAR AND WILLIAM D. DONACHIE" Institlute of Cell and Molecular Biology, University of Edinburgh, Darwin Building, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, Scotland Received 28 June 1993/Accepted 2 Septembcr 1993 A 490-bp DNA segment spanning the junction between the ftsa and ftsz genes inhibits cell division when present in high copy number. We show that this segment contains an antisense promoter and an antisense transcription terminator which define a new gene, stjz. High copy numbers of the 2.3-kbp ('ddlb) ftsq ftsa (fisz') chromosomal segment (Fig. 1) block cell division (5, 6, 8, 11). This inhibition of division has previously been ascribed to excess production of FtsA protein (5, 6, 8, 1 1). The block is to the earliest stage of division, that which requires FtsZ protein, and can be reversed by increasing the number of copies of the ftsz gene (4, 5). However, several observations have made us question whether the cause of the division block is excess FtsA protein alone. Firstly, T6tart and Bouch6 (10) have shown that the product of the dicf gene (a member of the dicb operon, apparently a relict phage operon, without known function in the cell cycle [1, 2, 7]) is an antisense RNA which is complementary to the sequence around the ribosomebinding site of ftsz mrna and that dicf RNA blocks translation of ftsz mrna. Secondly, as we have reported earlier (8), the 2.3-kbp chromosomal fragment contains within it a strong transcription terminator which, however, is oriented in such a way that it could terminate only transcription opposing that of ftsa or ftsz. A reverse RNA transcript of this segment would include an 8-base, GC-rich inverted repeat, separated by 10 bases and followed by an 19-base AU-rich sequence. This therefore resembles a rho-independent transcription terminator that, however, could operate to stop only reverse transcription (8) (Fig. 2). The location of this possible reverse terminator is such that reverse transcription of the ftsa-ftsz junction region starting from within ftsz would produce an antisense RNA that was complementary to the same mrna sequence as is dicf RNA. To test the idea that transcription from the antisense strand of ftsz, rather than overproduction of FtsA protein alone, is responsible for the inhibition of cell division by psz24, we removed the 1.8-kbp -HindlIl segment (containing the ftsq gene and most of ftsa) to produce psz25 (Fig. 1). We found that cells carrying psz25, like those carrying psz24 (5), are completely blocked in cell division at 42 C (Fig. 3), although the 490-bp chromosomal fragment remaining in this plasmid contains only the 3' end of theftsa gene and the 5' end of ftsz (Fig. 1). Therefore, excess FtsA protein cannot be the cause of the inhibition of cell division by psz25. To see whether the "reverse terminator" is within the 490-bp HindIll- fragment, as suggested by the DNA sequence (Fig. 2), we cloned this piece in both orientations between the gal promoter and the galk gene in the termination assay vector Corresponding author pjw30 (12) (to give psd55 and psd56 [Fig. 1]). Host cells with the galk mutation carrying the parent vector form red colonies on MacConkey-galactose plates. Colonies of cells with the galk mutation carrying P~,.1::[HindIII-]::galK+ also are red, but those carrying P.,,::[-HindIIIj::galK' are white. Therefore there is indeed a strong reverse transcription terminator that is able to block,,, transcription of this part of the ftsa-ftsz complementary strand. The same 490-bp piece of DNA was cloned in the orientation [-HindIII]::galK+ into the promoter assay vector, pkol. Colonies of host cells with the galk mutation carrying pns45 or pko are white on MacConkey-galactose plates (8), confirming that there is no promoter between the reverse terminator and the HindIll end of this piece of DNA. To test whether there is an internal reverse promoter at the end of this fragment, a 342-bp HindIII-Bsu36I section was removed to give pas 142. The Bsu36I restriction site is located within one of the inverted-repeat sequences of the proposed terminator (Fig. 2), and removal of the 342-bp fragment was therefore expected to abolish transcription termination. Colonies of host cells with the galk mutation carrying pas 142 are deep red on MacConkey-galactose plates. Restriction analysis confirmed the structure of pasl42 to be -Bsu361 ::galk+. The 148-bp -Bsu36I sequence therefore does indeed contain a strong reverse promoter. All the plasmids shown in Fig. 1 (except pas 142) contain the reverse promotcr and reverse terminator that together delimit a new antisense gene, stjz. However, of the plasmids containing intact stfz sequences, only psz24 and psz25 cause detectable inhibition of cell division (at 42 C). The reason that psz24 and psz25 inhibit division most strongly is probably because they have copy numbers of more than 200 per cell, whereas the remaining plasmids have copy numbers in the range of 20 to 50 per cell. Cells carrying pasl42 are also filamentous at 42 C, perhaps because this plasmid produces a hybrid stfz'::galk transcript which may fold into a configuration which more readily pairs with ftsz mrna than does stfz RNA itself. The experiments described here show that there are both a strong promoter and a strong transcription terminator within the 490-bp HindlIl- segment. This therefore defines a new antisense gene, stfz, which would be expected to produce an antisense RNA of not more than about 140 bases in length (allowing a minimum spacing of 40 bp between the site and the beginning of such an RNA and assuming that the

2 I ddlb ftsq ftsa ftsz 2290 HindIII Bsu36I stfz psz24 psz25 Plac L l pns30 L>iII galke1l pns45 (pko1-500) XI E psd55 > I> ---- I> -> r> C Pgal >> galk Gal + psd56 pas 142 IJ2E<II )IU2~I lv8'd + FIG. 1. The ddlb-to-ftsz section of the mra operon. The open reading frames of the four genes in this segment are shown as boxes, and at the top the scale is given in kilobase pairs. The positions of four restriction sites are shown, together with three subcloned segments (sizes in base pairs) relevant to this paper. Beneath this are shown the known promoters (arrowheads) and the deduced transcripts for this region. All sense transcripts are assumed to continue through ftsz and end at the terminator following enva (the next gene); the short antisense transcript (stfz) is assumed to end at the proposed antisense terminator (see text). Gal phenotypes of strains carrying the plasmids, are indicated at right. 7098

3 VOL. 175, 19'93 NOTES ftsA N%, 5' AAAAGAG1TITTAA ltt'tatgaggccgacgatgattacggcctcaggcgacaggcacaaatc...lys glu phe + Bsu36I ftsz... GGAAGGAAAC4 TGT'TGAACCAATGGAACTFACCAATGACGCGGTGAITAAAGTCATCGGC metpheglu... GTcGGIWCGG(GGGIGGTAATGCI?GTFGAACACATGGTGcGcGAGCGCATfGAAGGTGITTAAC 3' FIG. 2. 5'-to-3' sequence of theftsa-ftsz junction. A 60-bp stretch separates the stop codon offtsa from the start codon offtsz (vertical bars). The proposed "antiterminator" is the GC-rich 8-bp inverted repeat (horizontal arrows) preceded by the 19-bp AT-rich sequence. The putative ribosome-binding Shine-Dalgarno sequence for ftsz is underlined. The "antipromoter" must lie within the 148-bp Bsu36I- segment. Downloaded from transcript would end just beyond the inverted-repeat sequence). Our observations suggest that it is likely to be this transcript which is responsible for the inhibition of division in cells which carry this DNA segment in high copy number. Although it is attractive to suppose that the stjz reverse transcript acts, like dicf RNA, by hydrogen bonding to the ribosome-binding site offtsz mrna, it is nevertheless possible that stfz RNA is translated and that it is the translation product which inhibits cell division. There is one possible open reading frame within the region of the stfz RNA transcript. This is preceded by a possible Shine-Dalgarno motif and would code for a 25-amino-acid polypeptide. Further work is required to show whether this polypeptide is produced and whether it has a role in division control. We have shown that the segment of DNA which spans the region from the end offtsa through the 60-bp spacer sequence into the beginning of ftsz blocks cell division. We have also shown that this short segment contains both a strong reverse promoter and a strong reverse terminator, which should produce an antisense transcript of this region. However, a puzzling feature of both the dicf and stfz systems is that neither of them appears to be able to inhibit cell division except when greatly overexpressed at high temperature. A possible explanation for this is that each of these sense-antisense interactions requires the participation of a further factor, similar to the Rom-Rop protein which mediates the RNAI-RNAII hybridization of ColEl plasmids (3, 9). When either dicf RNA or stfz RNA alone is overproduced, there may not be enough of this second factor to allow hybridization or to stabilize the RNAs. Inhibition offtsz mrna translation would therefore take place to a significant extent only when either of the antisense RNAs is present in great excess and when the temperature is high enough to encourage melting of the secondary structure and hybridization with ftsz mrna. Future work will be directed to a search for this possible hybridization factor. Finally, we should emphasize that our present observations do not contradict earlier reports which showed that induction of excess FtsA protein also inhibits the activity of FtsZ (4, 6, 11). on December 19, 2018 by guest

4 7100 NOTES J. BA(UIIRI()I.. \ ibof ptz18r psz24 psz25 P w *W FIG. 3. Cells carrying ptzi8r (vector), psz24, or psz25 after growth overnight on nutrient agar plates at 42 C. Bar, 10,um.

5 VOL. 175, 1993 NOTES 7101 We thank K. J. Begg for the photomicrographs and M. Masters for discussing and reading the manuscript. This work is supported by a grant from the Wellcome Trust. REFERENCES 1. Bejar, S., and J.-P. Bouche A new dispensable genetic locus of the terminus region involved in control of cell division in Escherichia coli. Mol. Gen. Genet. 201: Bouche, F., and J.-P. Bouche Genetic evidence that DicF, a second division inhibitor encoded by the Escherichia coli dicb operon, is probably RNA. Mol. Microbiol. 3: Cesareni, G., M. Helmer-Citterich, and L. Castagnoli Control of ColEl plasmid replication by antisense RNA. Trends Genet. 7: Dai, K., and J. Lutkenhaus The proper ratio of FtsZ to FtsA is required for cell division to occur in Escherichia coli. J. Bacteriol. 174: Dewar, S. J., K. J. Begg, and W. D. Donachie Inhibition of cell division by an imbalance in the ratio of FtsA to FtsZ. J. Bacteriol. 174: Dewar, S. J., V. Kagan-Zur, K. J. Begg, and W. D. Donachie Transcriptional regulation of cell division genes in Escherichia coli. Mol. Microbiol. 3: Faubladier, M., K. Cam, and J.-P. Bouche Escherichia coli cell division inhibitor DicF-RNA of the dicb operon. Evidence for its generation in vivo by transcription termination and by RNaseIII and RNaseE-dependent processing. J. Mol. Biol. 212: Robinson, A. C., D. J. Kenan, G. F. Hatfull, N. F. Sullivan, R. Spiegelberg, and W. D. Donachie DNA sequence and transcriptional organization of essential cell division genes ftsq and ftsa of Escherichia coli: evidence for overlapping transcriptional units. J. Bacteriol. 160: Simons, R. W., and N. Kleckner Biological regulation by antisense RNA in prokaryotes. Annu. Rev. Genet. 22:567-60( Tetart, F., and J.-P. Bouche Regulation of the expression of the cell cycle geneftsz by DicF antisense RNA. Division does not require a fixed number of FtsZ molecules. Mol. Microbiol. 6: Wang, H., and R. C. Gayda High-level expression of the FtsA protein inhibits cell septation in Escherichia coli K-12. J. Bacteriol. 172: Wright, J. J., A. Kumar, and R. S. Hayward Hypersymmetry in a transcriptional terminator of Escherichia coli confers increased efficiency as well as bidirectionality. EMBO J. 11: Downloaded from on December 19, 2018 by guest